中华皮肤科杂志 ›› 2011, Vol. 44 ›› Issue (2): 117-120.

• 论著 • 上一篇    下一篇

以人羊膜为支架的表皮黑素细胞培养与生物活性鉴定

佘小光1,刘小明2,雷铁池2   

  1. 1. 湖北省武汉市武汉大学人民医院皮肤性病科
    2. 武汉大学人民医院
  • 收稿日期:2010-05-07 修回日期:2010-07-25 出版日期:2011-02-15 发布日期:2011-02-10
  • 通讯作者: 雷铁池 E-mail:tiechilei@gmail.com
  • 基金资助:

    多巴色素异构酶缺陷相关的氧化应激改变对黑素小体蛋白免疫原性影响研究

Culture of human epidermal melanocytes with human amniotic membrane as a scaffold and their biological ability identification

  • Received:2010-05-07 Revised:2010-07-25 Online:2011-02-15 Published:2011-02-10
  • Contact: Tie-Chi Lei E-mail:tiechilei@gmail.com

摘要:

目的 从吸引疱疱顶微小皮片建立人表皮黑素细胞(MC)原代培养,以人羊膜为支架培养MC,观察羊膜对MC黏附、增殖和树突发育的影响。方法 用负压吸疱法分离疱顶微小皮片,多巴染色后对取自同一个体下腹部和前臂屈侧皮片的MC进行计数。皮片经胰酶消化后自基底面轻刮表皮细胞建立原代MC培养,将培养的第2 ~ 4代MC接种至新鲜或冻存羊膜上共孵育不同时间后,倒置显微镜下观察MC形态和树突发育。行石蜡包埋组织切片,HE染色后观察MC对羊膜的黏附。用MTT比色法测定接种在羊膜上的MC存活率。结果 前臂屈侧吸引疱疱顶皮片多巴染色阳性细胞个数为(1543.1 ± 113.3)个/mm2,腹部皮片为(857.4 ± 101.7)个/mm2。在4周内建立MC原代培养至少需要从2个疱顶(面积约25.1 mm2)的前臂皮片获取的表皮细胞。倒置显微镜下观察,与单纯接种细胞培养皿的MC比较,接种至新鲜或冻存羊膜上继续培养4、8、12 d的MC形态多为两极,树突细长延伸。HE染色示MC能黏附并均匀分布在羊膜的基底膜层表面。MTT法测定结果示,羊膜对MC的分裂增殖有抑制,但冻存羊膜与新鲜羊膜对MC增殖抑制差异无统计学意义(P > 0.05)。结论 人前臂屈侧皮肤富含活性MC,是获取吸引疱疱顶微小皮片建立MC原代培养的较好部位。人羊膜支持MC黏附生长和树突发育,有望成为一种能荷载体外培养扩增MC的人源生物支架。

关键词: 白癜风

Abstract:

Objective To establish a primary culture of human melanocytes from tiny skin sheets harvested by using a suction blister method, to carry out a serial subcultivation of the melanocytes with human amniotic membrane (AM) as a scaffold, and to observe the influence of AM on the adhesion, proliferation and dendrite development of melanocytes. Methods Tiny skin sheets were collected from the flexual forearm or lower abdomen of a healthy male volunteer by a suction blister method and melanocytes in the skin sheet were counted following Dopa staining under a microscope. The trypsinized skin sheets were scraped with a scalpel to harvest melanocytes which were subjected to a primary culture. Then, the melanocytes were inoculated onto fresh or cryopreserved AM followed by a culture for various durations (4, 8 and 12 days). The morphology and dendrite development of melanocytes were visualized under an inverted microscope after dopa-staining, cell viability evaluated by MTT assay, the adhesion to AM examined by hematoxylin and eosin (HE) staining protocol. Results The density of melanocytes was 1543.1 ± 13.3 cells per mm2 and 857.4 ± 101.7 cells per mm2 in skin sheets obtained from the forearm flexure and lower abdomen of the volunteer, respectively. A skin sheet of about 25.1 mm2 from approximately two blister roof was required to ensure the success of primary culture of melanocytes within 1 month. After culture on fresh or cryopreserved AM for 4, 8, and 12 days, most melanocytes were bi-polar with extended slender dendrites compared with those cultured in common cell culture medium. HE staining showed that melancytes adhered and were evenly distributed on the basement membrane of AM. MTT assay showed that the AM inhibited the proliferation of melanocytes, and no statistical difference was observed in the inhibitory effect between fresh AM and cryopreserved AM (P > 0.05). Conclusions Enriched with melanocyes, flexural forearm is a preferable donor site to offer skin sheets for primary culture of melanocytes. Human AM could improve the adhesive growth and dendrite development of melanocytes, and may serve as a promising bioscaffold for in vitro expansion of melanocytes.

Key words: vitligo